Modulation of complement-dependent cytotoxicity through modifications of the C-terminus of antibody heavy chains

ABSTRACT

Antibody variants having decreased or increased ability to mediate CDC due to modifications at the C-terminus of their heavy chains are described. Methods of generating such antibodies, as well as nucleotide constructs and host cells suitable for the production of said antibodies are also described.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/130,580, filed on Apr. 21, 2014, which is a 35 U.S.C. 371 nationalstage filing of PCT/EP2012/063338, filed on Jul. 6, 2012, which claimsthe benefit of U.S. Provisional Application No. 61/504,987, filed onJul. 6, 2011, and Danish Patent Application No. PA 2011 00518, filed onJul. 6, 2011. The contents of the aforementioned applications are herebyincorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 30, 2019, isnamed GMI_136USDV_Sequence_Listing.txt and is 15,579 bytes in size.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/130,580, filed on Apr. 21, 2014 (now U.S. Pat. No. 10,323,081), whichis a 35 U.S.C. 371 national stage filing of PCT/EP2012/063338, filed onJul. 6, 2012, which claims the benefit of U.S. Provisional ApplicationNo. 61/504,987, filed on Jul. 6, 2011, and Danish Patent Application No.PA 2011 00518, filed on Jul. 6, 2011. The contents of the aforementionedapplications are hereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 30, 2019, isnamed GMI_136USDV_Sequence_Listing.txt and is 15,579 bytes in size.

FIELD OF THE INVENTION

The present invention relates to antibodies and their ability to inducecomplement-dependent cytotoxicity (CDC). Antibody variants are providedthat have altered ability to mediate CDC due to modifications at theC-terminus of their heavy chains. Furthermore, the invention providesmethods of generating such antibodies, and nucleotide constructs andhost cells suitable for the production of said antibodies.

BACKGROUND OF THE INVENTION

Monoclonal antibodies have in recent years become successful therapeuticmolecules, in particular for the treatment of cancer and autoimmunediseases. Effector functions mediated by the Fc region of antibodies,such as antibody-dependent cell-mediated cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC) are often important mechanismsfor the clinical efficacy of monoclonal antibodies.

Antibody-induced CDC is mediated through the proteins of the classicalcomplement cascade. This cascade is triggered by binding of thecomplement protein C1q to the antibody. C1q is composed of a bundle ofsix heterotrimeric subunits having globular heads and collagen-liketails. Binding of C1q to the Fc region of an antibody is known toinvolve the CH2 region of the antibody (Duncan and Winter (1988) Nature332:738). Furthermore, sugar moieties on the Fc region are known toinfluence the binding of C1q (Raju (2008) Curr Opin Immunol 20:471).

Monoclonal antibodies are complex molecules that can undergo many typesof enzymatic and non-enzymatic post-translational modifications,including formation of disulfide bonds, glycosylation, glycation,N-terminal glutamine cyclization, C-terminal lysine processing(removal), deamidation, isomerization, oxidation and peptide bondcleavage (for a review of these modifications, see by Liu et al. (2008)J Pharm Sci 97: 2426).

Removal of C-terminal lysines by carboxypeptidases from the heavy chainis a commonly observed antibody modification, both upon recombinantexpression of antibodies in mammalian cells, as well as in vivo in humanserum (Cai et al. (2010) Biotechnol. Bioeng. September 9). Removal isoften partial, resulting in a mixed population of antibodies with zero(K0), one (K1) or two (K2) C-terminal lysines (i.e., in the case of K2,one C-terminal lysine in each heavy chain of the antibody). Inparticular, B cell hybridomas produce mixtures of K0, K1 and K2molecules (Dick et al. (2008) Biotech. Bioeng. 100:1132).

WO 2009027471 describes that deletion of the codon for the C-terminallysine of a heavy chain of an antibody can result in higher antibodytiters upon expression in Chinese Hamster Ovary (CHO) cells.

WO 2008006554 describes that an antibody preparation produced inΔxyl-t/Δfuc-t moss cells comprises an N-glycan structure free of fucoseand xylose and lacks C-terminal lysine residues. The ability of thispreparation to mediate ADCC was less inhibited by serum than apreparation of the same antibody produced in murine Sp2/0 cells.Complement-mediated lytic activity, on the other hand, was reduced.Removal of the C-terminal lysine residue was proposed to increase ADCC,although the effect of this modification alone was not tested.

The general belief in the art has been that C-terminal lysines havelittle or no effect on antibody function, see e.g. the literature reviewby Cai et al. (2010) supra, which concluded that no activity had beenattributed to C-terminal lysines, or Harris (2005) Dev Biol (Basel)122:127 which stated that the presence or absence of heavy chain Lysresidues had no effect on antigen binding, and was not likely toinfluence Fc effector functions, clearance or any other biologicalproperty.

Antes et al. (2007) J Chromatogr. B 852:250 described testing the effectof C-terminal lysines on CDC. Antibody preparations were generated inSp2/0 cells containing a significant subpopulation of C-terminallysine-containing antibody molecules. Proteolytic removal of theC-terminal lysines had no effect on the ability of these antibodypreparations to mediate CDC. The authors concluded, e.g., that bothantibody variants—clipped and unclipped—elicited the same potency in acomplement dependent cytotoxicity (CDC) assay demonstrating that lysineclipping of IGN311 does not impair Fc-mediated effector functions.

SUMMARY OF THE INVENTION

It has now surprisingly been found that lysines and other charged aminoacid residues at the C-terminus of the heavy chains of an antibody do infact have a major impact on their ability to mediate CDC.

Without being bound by any specific theory, it is hypothesized that thiswas not detected previously, because pure preparations of the K0, K1 andK2 forms were not compared.

The observation, described in the Examples herein, of the relationshipbetween the presence of C-terminal charged residues and potency tomediate CDC provides the basis for novel antibody products havingaltered CDC properties as well as novel antibody production methods.

In a first aspect, the present invention provides a method for producingan antibody in a host cell, such as an antibody having increased CDC,wherein said antibody comprises at least a heavy chain, said methodcomprising the following steps:

a) providing a nucleotide construct encoding said heavy chain, whereinsaid construct does not encode a lysine residue at the C-terminus ofsaid heavy chain,

b) expressing said nucleotide construct in a host cell, with the provisothat said host cell is not a CHO cell or a moss cell, and

c) recovering said antibody from a cell culture of said host cell.

In another aspect, the invention relates to a method for increasing theability of an antibody to mediate CDC, said method comprising the stepof removing a C-terminal lysine residue from the heavy chains of anantibody.

The product of the above methods is a population of antibody moleculeshaving an increased ability to mediate CDC as compared to correspondingantibody molecules containing a C-terminal lysine residue. Suchantibodies are useful for a number of purposes, e.g. therapeuticpurposes, where killing of target cells is desired. This may, forexample, be desired in the treatment of cancer.

In a further aspect, the invention provides an antibody variantcomprising a heavy chain comprising at least one charged amino acidresidue in the C-terminal region, wherein said charged amino acidresidue is not susceptible to proteolytic removal.

This antibody variant has a decreased ability to mediate CDC as comparedto a corresponding antibody molecule lacking one or both C-terminallysine residues. Such antibodies are useful for applications, e.g.therapeutic applications, where only silencing of a target is desired.This can be advantageous in, e.g., the treatment of autoimmune diseasesor inflammation.

In further aspects, the invention relates to methods, nucleotideconstructs and host cells for producing antibody variants having atleast one charged amino acid residue in the C-terminal region of theheavy chain, wherein said charged amino acid residue is not susceptibleto proteolytic removal.

In a further aspect, the invention relates to a mammalian cell which hasbeen genetically modified to eliminate the activity of carboxypeptidasescapable of removing C-terminal lysines from heavy chains.

In even further aspects, the invention provides compositions ofantibodies which have been modified to strengthen intermolecularinteractions between antibody molecules in order to strengthen theirability to mediate effector functions, such as CDC. In one such aspect,the invention relates to an antibody mixture comprising a subpopulationof antibody variant molecules comprising heavy chains having at leastone positively-charged amino acid residue in the C-terminal region and asubpopulation of antibody variant molecules comprising heavy chainshaving at least one negatively-charged amino acid residue in theC-terminal region, wherein said positively-charged andnegatively-charged amino acid residues are not susceptible toproteolytic removal.

In another such aspect, the invention provides an antibody comprising aheavy chain having a variant C-terminal region, comprising one or moreamino acid modifications that favour intermolecular C-terminalinteractions between antibody molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F: CIEX profiles, cIEF and SDS-PAGE analysis of C-terminallysine isoforms of anti-CD20 and anti-CD38 antibodies.

CIEX profiles of anti-CD20 (FIG. 1A) and anti-CD38 (FIG. 1B) antibodies.cIEF profiles of unfractionated antibody (unfr.), and collectedisoforms, containing zero, one or two C-terminal lysines per IgGmolecule (K0, K1, K2), of anti-CD20 (FIG. 1C) and anti-CD38 (FIG. 1D)antibodies. SDS PAGE analysis of unfractionated antibodies and collectedisoforms of anti-CD20 (FIG. 1E) and anti-CD38 (FIG. 1F) antibodies.Samples were analyzed untreated (−) or after carboxypeptidase Btreatment (+) (FIGS. 1C-1F).

FIG. 2 —Binding curves and CDC induction by C-terminal lysine isoformsof anti-CD20 and anti-CD38 antibodies.

Binding to Daudi cells of unfractionated antibodies (unfr.) andcollected K2 isoforms of anti-CD20 and anti-CD38 antibodies with andwithout carboxypeptidase B (CPB) treatment, as determined by FACSanalysis (upper panel). Data shown are mean fluorescence intensities(MFI). Induction of CDC of Daudi cells by unfractionated antibodies andcollected K2 isoforms of anti-CD20 and anti-CD38 antibodies with andwithout carboxypeptidase B treatment, as measured using propidium iodidestaining method (lower panel). Data shown are percentages lysis.

FIG. 3 —Efficacy of C1q utilization by C-terminal lysine isoforms ofanti-CD38 antibody.

Induction of CDC of Daudi cells by unfractionated and collected K2isoform of anti-CD38 antibody with and without carboxypeptidase B (CPB)treatment, as measured using propidium iodide staining method. Cellswere incubated with a fixed antibody concentration (10 μg/mL). Titratedamounts of C1q were added in the assay as complement source. Data shownare mean percentages of lysis±S.E.M of three experiments.

FIGS. 4A-4C—cIEF analysis, binding and CDC induction by anti-CD38antibody and mutants.

cIEF analysis of anti-CD38 antibody and mutants (FIG. 4A). Binding ofanti-CD38 antibody and mutants to Daudi cells as analyzed by FACSanalysis (FIG. 4B). Data shown are mean fluorescence intensities (MFI).Induction of CDC of Daudi cells by anti-CD38 antibody and mutants asmeasured by PI staining method (FIG. 4C). Data shown are percentageslysis.

FIGS. 5A-5D—CDC induction by mixtures of anti-CD38 antibody mutants.

K0 and K4 (FIG. 5A) or K0 and E2 (FIG. 5B) mutants were mixed indifferent ratios, as indicated, and induction of CDC of Daudi cells wasmeasured by propidium iodide staining method. Total IgG concentrationwas 10 μg/mL. Induction of CDC by K4 mutant, E2 mutant or a 1:1 mixtureof both (FIG. 5C). Total IgG concentration was titrated from 10 μg/mL to0.016 μg/mL. K0 is shown as positive control. Induction of CDC by E2:K4mutant mixtures in different ratios, at two total IgG concentrations (10μg/mL and 0.25 μg/mL) (FIG. 5D). Data shown (FIGS. 5A-5D) arepercentages (maximal) lysis.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “immunoglobulin” refers to a class of structurally relatedglycoproteins consisting of two pairs of polypeptide chains, one pair oflight (L) low molecular weight chains and one pair of heavy (H) chains,all four inter-connected by disulfide bonds. The structure ofimmunoglobulins has been well characterized. See for instanceFundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)). Briefly, each heavy chain typically is comprised of a heavychain variable region (abbreviated herein as VH) and a heavy chainconstant region. The heavy chain constant region typically is comprisedof three domains, CH1, CH2, and CH3. The heavy chains areinter-connected via disulfide bonds in the so-called “hinge region”.Each light chain typically is comprised of a light chain variable region(abbreviated herein as VL) and a light chain constant region. The lightchain constant region typically is comprised of one domain, CL.Typically, the numbering of amino acid residues in the constant regionis performed according to the EU-index as described in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). The VH andVL regions may be further subdivided into regions of hypervariability(or hypervariable regions which may be hypervariable in sequence and/orform of structurally defined loops), also termed complementaritydetermining regions (CDRs), interspersed with regions that are moreconserved, termed framework regions (FRs). Each VH and VL is typicallycomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901 917 (1987)).

The term “antibody” (Ab) in the context of the present invention refersto an immunoglobulin molecule, a fragment of an immunoglobulin molecule,or a derivative of either thereof, which has the ability to specificallybind to an antigen under typical physiological conditions with a halflife of significant periods of time, such as at least about 30 minutes,at least about 45 minutes, at least about one hour, at least about twohours, at least about four hours, at least about eight hours, at leastabout 12 hours, about 24 hours or more, about 48 hours or more, aboutthree, four, five, six, seven or more days, etc., or any other relevantfunctionally-defined period (such as a time sufficient to induce,promote, enhance, and/or modulate a physiological response associatedwith antibody binding to the antigen and/or time sufficient for theantibody to recruit an effector activity). The variable regions of theheavy and light chains of the immunoglobulin molecule contain a bindingdomain that interacts with an antigen. The constant regions of theantibodies may mediate the binding of the immunoglobulin to host tissuesor factors, including various cells of the immune system (such aseffector cells) and components of the complement system such as C1q, thefirst component in the classical pathway of complement activation. Anantibody may also be a bispecific antibody, diabody or similar molecule.The term “bispecific antibody” refers to antibody having specificitiesfor at least two different, typically non-overlapping, epitopes. Asindicated above, the term antibody herein, unless otherwise stated orclearly contradicted by the context, includes fragments of an antibodythat retain the ability to specifically bind to the antigen. Suchfragments may be provided by any known technique, such as enzymaticcleavage, peptide synthesis and recombinant expression techniques. Ithas been shown that the antigen-binding function of an antibody may beperformed by fragments of a full-length antibody. It also should beunderstood that the term antibody, unless specified otherwise, alsoincludes polyclonal antibodies, monoclonal antibodies and antibody-likepolypeptides, such as chimeric antibodies and humanized antibodies. Anantibody as generated can possess any isotype. In one embodiment of theinvention, the antibody is isolated and/or purified.

As used herein, “isotype” refers to the immunoglobulin class (forinstance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that is encodedby heavy chain constant region genes.

Unless stated otherwise, the term “C-terminal lysine residue” refers tothe lysine residue at the C-terminus of the heavy chain (i.e. theC-terminus of the CH3 region).

Unless stated otherwise, the term “C-terminal region”, refers to theC-terminal region of the heavy chain of an antibody, consisting of thesix most C-terminal amino acids of the heavy chain polypeptide.

The term “full-length antibody” when used herein, refers to an antibodywhich contains all heavy and light chain constant and variable domainsthat are normally found in an antibody of that isotype.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

When used herein, the term “heavy-chain antibody” refers to an antibodywhich consists only of two heavy chains and lacks the two light chainsusually found in antibodies. Heavy-chain antibodies, which naturallyoccur in e.g. camelids, can bind antigens despite having only VHdomains, see e.g. Hamers-Casterman (1993) Nature 363:446.

As used herein, the term “binding”, in the context of the binding of anantibody to a predetermined antigen, typically is a binding with anaffinity corresponding to a K_(D) of about 10⁻⁶ M or less, e.g. 10⁻⁷ Mor less, such as about 10⁻⁸ M or less, such as about 10⁻⁹ M or less,about 10⁻¹⁰ M or less, or about 10⁻¹¹ M or even less, when determined byfor instance surface plasmon resonance (SPR) technology in a BIAcore3000 instrument, using the antigen as the ligand and the antibody as theanalyte, and binds to the predetermined antigen with an affinitycorresponding to a K_(D) that is at least ten-fold lower, such as atleast 100-fold lower, for instance at least 1,000-fold lower, such as atleast 10,000-fold lower, for instance at least 100,000-fold lower thanits affinity for binding to a non-specific antigen (e.g., BSA, casein),other than the predetermined antigen or a closely-related antigen. Theamount with which the affinity is lower is dependent on the K_(D) of theantibody, so that when the K_(D) of the antibody is very low (that is,the antibody is highly specific), then the amount with which theaffinity for the antigen is lower than the affinity for a non-specificantigen may be at least 10,000-fold. The term “K_(D)” (M), as usedherein, refers to the dissociation equilibrium constant of a particularantibody-antigen interaction.

An “isolated antibody,” as used herein, denotes that the material hasbeen removed from its original environment (e.g., the naturalenvironment if it is naturally occurring or the host cell if it isrecombinantly expressed). It is also advantageous that the antibodies bein purified form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, indicating an increaseof the antibody concentration relative to the concentration ofcontaminants in a composition as compared to the starting material.

The term “host cell”, as used herein, is intended to refer to a cellinto which a recombinant nucleotide construct or an expression vectorhas been introduced, e.g. a nucleotide construct or an expression vectorencoding an antibody of the invention. Recombinant host cells include,for example, transfectomas, such as CHO cells, HEK293 cells, NSO cellsor lymphocytic cells, or plant cells, fungal cells, such as yeast cellsor prokaryotic cells.

Further Aspects and Embodiments of the Invention

As described above, in a first aspect, the invention relates to methodsfor increasing the ability of an antibody to mediate CDC by reducing thenumber of charged amino acid residues in the C-terminal region of theheavy chain. Antibody products having increased ability to mediate CDCcan be obtained by post-translational proteolytic removal of the chargedresidue from the heavy chain or by recombinant production of theantibody from a nucleotide construct lacking codons for charged aminoacids, such as lysines, in the region encoding the C-terminus.

Thus, in a first aspect, the invention provides a method for producingan antibody in a host cell, wherein said antibody comprises at least aheavy chain, said method comprising the following steps:

-   -   a) providing a nucleotide construct encoding said heavy chain,        wherein said construct does not encode a lysine residue at the        C-terminus of said heavy chain,    -   b) expressing said nucleotide construct in a host cell, with the        proviso that said host cell is not a CHO cell or a moss cell,        and    -   c) recovering said antibody from a cell culture of said host        cell.

Typically, the heavy chain comprises a constant region comprising atleast a CH3 domain, at least a CH2 and a CH3 domain, or all of a CH1, aCH2 and a CH3 domain. In one embodiment, the heavy chain comprises atleast a CH2 and a CH3 domain.

In some embodiments, the antibody is a heavy-chain antibody. In mostembodiments, however, the antibody will also contain a light chain andthus said host cell further expresses a light-chain-encoding construct,either on the same or a different vector.

Host cells suitable for the recombinant expression of antibodies arewell-known in the art. In a one embodiment, said host cell is a cellwhich is capable of Asn-linked glycosylation of proteins, e.g. aeukaryotic cell, such as a mammalian cell, e.g. a human cell. In afurther embodiment, said host cell is a non-human cell which isgenetically engineered to produce glycoproteins having human-like orhuman glycosylation. Examples of such cells are genetically-modifiedPichia pastoris [Hamilton et al., Science 301 (2003) 1244-1246;Potgieter et al., J. Biotechnology 139 (2009) 318-325] andgenetically-modified Lemna minor [Cox et al., Nature Biotechnology 12(2006) 1591-1597].

In one embodiment, said host cell is not capable of efficiently removingC-terminal lysine residues from antibody heavy chains. For example,Table 2 in Liu et al. (2008) J Pharm Sci 97: 2426 (incorporated hereinby reference) lists a number of such antibody production systems, e.g.Sp2/0, NS0 or transgenic mammary gland (goat), wherein only partialremoval of C-terminal lysines is obtained.

More specifically, such a host cell may be a cell which, when expressinga C-terminal lysine-containing construct, would produce an antibodypreparation wherein more than 10% of the antibody molecules is in the K2isoform, e.g. wherein more the 30% is in the K2 isoform. Table 1 givesexpected amounts of K0, K1 and K2 as a function of C-terminal cleavage.Thus, phrased in another manner, a preferred host cell is a host cell inwhich more than 30% of the heavy chains remain uncleaved at theC-terminus, e.g. wherein more than 60% of the heavy chains remainuncleaved at the C-terminus of the heavy chain.

TABLE 1 % uncleaved % K0 in batch % K1 in batch % K2 in batch 10 81 18 120 64 32 4 30 49 42 9 40 36 48 16 50 25 50 25 60 16 48 36 70 9 42 49 804 32 64 90 1 18 81 100 0 0 100

In a further embodiment of the invention, said host cell is selectedfrom the group consisting of:

-   -   (i) a yeast cell, e.g. a Pichia pastoris or Saccharomyces        cerevisiae, Hansenula polymorpha and Ogataea minuta and    -   (ii) a filamentous fungal cell, such as Aspergillus awamori,        Aspergillus niger, Aspergillus oryzea, Trichoderma reesi and    -   (iii) a plant cell, such as an Arabidopsis cell, Lemna minor,        Nicotiana benthamiana (tobacco), oilseed crop plant (Brassica        napus), soybean, rice, maize (Zea mays) or carrot cells, and    -   (iv) an NS0, Sp2/0 or PER.C6 cell.

Recombinant production of antibodies in yeasts and filamentous fungi hase.g. been described and reviewed by Joosten et al. (2003) Microbial CellFactories 2:1 and Gasser and Mattanovich (2007) Biotech Lett 29:201. Forproduction of antibodies in plant cells, see e.g. Cox et al. (2006) NatBiotechnol 24:1591, Decker and Reski (2007) Curr Opin Biotechnol 18:393,Giritch et al. (2006) Proc Natl Acad Sc USA 103:14701 or Hood et al.(2002) Curr Opin Biotechnol 13:630. Antibody expression in NSO celllines has, for example, been described by Dempsey et al. (2003)Biotechnol. Prog. 19:175. Jones et al. (2003) Biotechnol Prog 19:163,inter alia, have described antibody production in human PER.C6 cells.For antibody production in Sp2/0 cells, see e.g. Yang et al. (2007)Biotechnol Bioeng. 98:141.

In a further embodiment, wherein said heavy-chain-encoding constructdoes not encode a lysine or an arginine at the C-terminus of said heavychain, preferably said heavy-chain-encoding construct does not encode acharged amino acid residue at the C-terminus of said heavy chain. Morepreferably, said heavy-chain-encoding construct does not contain anycodon for a charged amino acid residue among the codon coding for thesix most C-terminal amino acids.

In one embodiment, the antibody is a human antibody comprising a humanheavy chain. The antibody can be of any isotype, including, but notlimited to, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, and IgM. In oneembodiment, the antibody is of an IgG isotype, such as an isotypeselected from IgG1, IgG2, IgG3, and optionally IgG4. Specific wild-typeheavy-chain constant sequences are provided below in SEQ ID NOS:1-5.However, different allotypes of each isotype are known in the art, andare described in Jefferies and Lefranc (2009), mAbs 1:4, 1-7, herebyincorporated by reference in its entirety.

In a further embodiment of the above method of the invention, thenucleotide construct provided in step a) is derived from, or designedbased on, an original heavy-chain sequence having a codon for aC-terminal lysine residue. For example, the original heavy-chainsequence may comprise a C-terminal sequence which is residues 325-330 ofSEQ ID NOS:1 (IgG1m (az) allotype) or 5 (IgG1m(f) allotype), or residues321 to 326 of SEQ ID NO:4, as set forth below. Thus, said nucleotideconstruct may comprise a deletion or a substitution of the codon for theC-terminal lysine (k) residue compared to said original heavy-chainsequence. Accordingly, in one embodiment, the nucleotide constructcomprises a C-terminal sequence which comprises or consists of residues325-329 of SEQ ID NOS:1 or 5. In one embodiment, the nucleotideconstruct comprises a C-terminal sequence which comprises or consists ofresidues 321 to 325 of SEQ ID NO:4.

In one embodiment, said nucleotide construct encodes a heavy chaincomprising a constant region sequence which, other than the C-terminalmodification, comprises at least a CH3 domain, at least CH2 and CH3domains, or CH1, CH2 and CH3 domains of the IgG1 isotype, optionally theIgG1m (f) allotype. In one embodiment, said nucleotide construct encodesa heavy chain comprising a constant region sequence which comprises atleast a CH3 domain, at least CH2 and CH3 domains, or CH1, CH2 and CH3domains of the IgG2 isotype, other than the C-terminal modification. Inone embodiment, said nucleotide construct encodes a heavy chaincomprising a constant region sequence which comprises at least a CH3domain, at least CH2 and CH3 domains, or CH1, CH2 and CH3 domains of theIgG3 isotype, other than the C-terminal modification. In one embodiment,said nucleotide construct encodes a heavy chain comprising a constantregion sequence which comprises at least a CH3 domain, at least CH2 andCH3 domains, or CH1, CH2 and CH3 domains of the IgG4 isotype, other thanthe C-terminal modification. In one embodiment, the heavy chaincomprises a sequence selected from SEQ ID NOS:1-5, apart from theC-terminal lysine residue.

In an even further embodiment, said nucleotide construct, other than theC-terminal modification, encodes a heavy chain of the IgG1 or IgG2isotype, i.e. has a constant region sequence which is identical insequence to the IgG1 or IgG2 constant region, except for the C-terminalmodification. In one embodiment, said nucleotide construct, other thanthe C-terminal modification, encodes a heavy chain of the IgG1 or IgG3isotype, i.e. has a constant region sequence which is identical insequence to the IgG1 or IgG3 constant region, except for the C-terminalmodification. In a further embodiment, the C-terminal codon of saidnucleotide construct encodes a Pro or a Gly residue, e.g. the-Pro-Gly-Lys sequence at the C-terminus of an IgG1 or IgG2 heavy chainwould be truncated to -Pro-Gly or -Pro only.

In a further aspect, the invention relates to an antibody obtained orobtainable by the method of the invention described above.

In a yet further aspect, the invention relates to a host cell which isnot capable of efficiently removing C-terminal lysine residues fromantibody heavy chains, said host cell comprising a nucleotide constructencoding a heavy chain lacking a C-terminal lysine. Preferably, saidhost cell is not moss or CHO. More preferably, said host cell is of oneof the types specified above under (i) to (iv).

In an even further aspect, the invention relates to a method forincreasing the ability of an antibody to mediate CDC, said methodcomprising the step of removing a C-terminal lysine residue from theheavy chains of an antibody. In a preferred embodiment, the lysineresidue is removed by enzymatic cleavage, e.g. using a carboxypeptidase,such as carboxypeptidase B or carboxypeptidase N (Cia et al. (2010)Supra). In one embodiment, said antibody is purified prior to removal ofthe C-terminal lysine residue.

As mentioned above, some cell types, e.g. hybridomas, produceheterogenous populations of antibody molecules, wherein some moleculescontain one or two C-terminal lysines and others do not. As it is clearfrom the present work that C-terminal lysines affect the ability ofantibodies to mediate CDC, such C-terminal heterogeneity may beundesired if different antibodies from a plurality of samples are to becompared, as there may be differences between different hybridomas withrespect to the degree of C-terminal processing. To eliminate suchdifferences, it may be advantageous to remove the C-terminal lysinesfrom antibody samples prior to comparative testing in order to be ableto better compare properties related to the binding properties of theantibodies.

Accordingly, in a further aspect, the invention relates to a method fortesting a plurality of antibody-producing cell cultures, e.g. hybridomacell cultures, for a desired property, said method comprising treatingsamples of said cultures to remove the C-terminal lysines as describedabove and testing the treated samples for the desired property. In someembodiments, it may be advantageous to remove cells and/or purifyantibody from the culture sample before removal of the C-terminallysines. In a preferred embodiment, the desired property to be tested iscell lysis or cell killing.

As explained above, in a further aspect, the invention relates to anantibody variant comprising a heavy chain having at least one chargedamino acid residue in the C-terminal region, such as one, two, three,four or more charged amino acids, wherein said charged amino acidresidue is not susceptible to proteolytic removal.

Examples of charged amino acids residues include those having a positivecharge, such as lysine (K or Lys), arginine (R or Arg) and histidine (Hor His), and those having a negative charge, such as glutamic acid (E orGlu) and aspartic acid (D or Asp). In one embodiment, the charged aminoacid residue is selected from lysine, arginine and histidine, such asfrom lysine and arginine, such as lysine. In one embodiment, the chargedamino acid residue is selected from glutamic acid and aspartic acid,such as glutamine.

Such variants typically have reduced ability to mediate CDC as comparedto a corresponding antibody lacking the charged residue. Such variantsmay e.g. be useful for therapeutic uses wherein reduced CDC is desired,e.g. uses wherein silencing of a target antigen, without killing of thetarget cell, is desired.

In some embodiments, the antibody is a heavy-chain antibody. In mostembodiments, however, the antibody will further comprise a light chain.

In a one embodiment, said charged amino acid residue is not susceptibleto removal by proteases of mammalian origin, preferably not susceptibleto removal by proteases of CHO, HEK-293, PER.C6, NS0 or Sp2/0 origin,more preferably not susceptible to removal by proteases, such ascarboxypeptidases, active in the secretory pathway of CHO, HEK-293,PER.C6, NS0 or Sp2/0 cells.

In a further embodiment, said charged amino acid residue is notsusceptible to removal by human proteases, preferably not susceptible toremoval by human proteases that can act on proteins in circulation, e.g.in blood.

In one embodiment, said charged amino acid is amongst the six mostC-terminal amino acid residues, e.g. amongst the five most C-terminalamino acid residues of said heavy chain, such as amongst the four mostC-terminal amino acid residues of said heavy chain, e.g. amongst thethree most C-terminal amino acid residues of said heavy chain, such asamongst the two most C-terminal amino acid residues of said heavy chain,e.g. wherein said charged amino acid is the most C-terminal amino acidresidue of said heavy chain.

In a further embodiment, said heavy chain comprises the CH3 sequence setforth in SEQ ID NO:1, 2, 3, 4 or 5 herein below, modified such that saidcharged amino acid is situated amongst the most six most C-terminalamino acid residues, i.e. from position 325 to 330, from position 321 to326, from position 372 to 377, from 321 to 326 or from position 325 to330, respectively.

SEQ ID NO: 1: The amino acid sequence of the IgG1 constant region(accession number P01857; IgGm(za) allotype) (the CH3 sequence isunderlined)   1 astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv 51 htfpavlqss glyslssvvt vpssslgtqt yicnvnhkps ntkvdkkvep 101kscdkthtcp pcpapellgg psvflfppkp kdtlmisrtp evtcvvvdvs 151hedpevkfnw yvdgvevhna ktkpreeqyn styrvvsvlt vlhqdwlngk 201eykckvsnka lpapiektis kakgqprepq vytlppsrde ltknqvsltc 251lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw 301qqgnvfscsv mhealhnhyt qkslslspgkSEQ ID NO: 2: The amino acid sequence of the IgG2 constant region(accession number P01859) (the CH3 sequence is underlined)   1astkgpsvfp lapcsrstse staalgclvk dyfpepvtvs wnsgaltsgv  51htfpavlqss glyslssvvt vpssnfgtqt ytcnvdhkps ntkvdktver 101kccvecppcp appvagpsvf lfppkpkdtl misrtpevtc vvvdvshedp 151evqfnwyvdg vevhnaktkp reeqfnstfr vvsvltvvhq dwlngkeykc 201kvsnkglpap iektisktkg qprepqvytl ppsreemtkn qvsltclvkg 251fypsdiavew esngqpenny kttppmldsd gsfflysklt vdksrwqqgn 301vfscsvmhea lhnhytqksl slspgkSEQ ID NO: 3: The amino acid sequence of the IgG3 constant region(accession number P1860) (the CH3 sequence is underlined)   1astkgpsvfp lapcsrstsg gtaalgclvk dyfpepvtvs wnsgaltsgv  51htfpavlqss glyslssvvt vpssslgtqt ytcnvnhkps ntkvdkrvel 101ktplgdttht cprcpepksc dtpppcprcp epkscdtppp cprcpepksc 151dtpppcprcp apellggpsv flfppkpkdt lmisrtpevt cvvvdvshed 201pevqfkwyvd gvevhnaktk preeqynstf rvvsvltvlh qdwlngkeyk 251ckvsnkalpa piektisktk gqprepqvyt lppsreemtk nqvsltclvk 301gfypsdiave wessgqpenn ynttppmlds dgsfflyskl tvdksrwqqg 351nifscsvmhe alhnrftqks lslspgkSEQ ID NO: 4: The amino acid sequence of the IgG4 constant region (theCH3 sequence is underlined)astkgpsvfp lapcsrstse staalgclvk dyfpepvtvs wnsgaltsgvhtfpavlqss glyslssvvt vpssslgtkt ytcnvdhkps ntkvdkrveskygppcpscp apeflggpsv flfppkpkdt lmisrtpevt cvvvdvsqedpevqfnwyvd gvevhnaktk preeqfnsty rvvsvltvlh qdwlngkeykckvsnkglps siektiskak gqprepqvyt lppsqeemtk nqvsltclvkgfypsdiave wesngqpenn ykttppvlds dgsfflysrl tvdksrwqegnvfscsvmhe alhnhytqks lslslgkSEQ ID NO: 5: The amino acid sequence of the IgG1m(f) allotype constantregion (the CH3 sequence is underlined)   1astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv  51htfpavlqss glyslssvvt vpssslgtqt yicnvnhkps ntkvdkrvep 101kscdkthtcp pcpapellgg psvflfppkp kdtlmisrtp evtcvvvdvs 151hedpevkfnw yvdgvevhna ktkpreeqyn styrvvsvlt vlhqdwlngk 201eykckvsnka lpapiektis kakgqprepq vytlppsree mtknqvsltc 251lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw 301qqgnvfscsv mhealhnhyt qkslslspgk

In a further embodiment, said heavy chain comprises the entire constantregion sequence set forth in SEQ ID NO:1, 2, 3, 4 or 5 and said chargedamino acid is situated amongst the six most C-terminal amino acidresidues, i.e. from position 325 to 330, from position 321 to 326 orfrom position 372 to 377, respectively.

In one embodiment, said charged amino acid is a positively charged aminoacid residue, preferably a lysine residue. As C-terminal lysines arenormally susceptible to proteolytic removal, said variant will containone or more amino acid modifications, such as additions orsubstitutions, at a position situated C-terminally of said lysine. Forexample, in one embodiment, said charged amino acid residue is notsusceptible to proteolytic removal due to the presence of a prolineresidue situated C-terminally of said charged amino acid residue,wherein said proline residue is preferably placed immediately C-terminalof said charged amino acid residue, more preferably wherein said chargedamino acid residue and said proline residue are the two most C-terminalamino acid residues of said heavy chain. For example, the antibody heavychain may be an IgG1, IgG2, IgG3 or IgG4 heavy chain to which a prolineresidue has been added at the C-terminus.

In another embodiment, said charged amino acid is a negatively-chargedamino acid residue, preferably a glutamic acid residue.Negatively-charged C-terminal residues are normally not susceptible toproteolytic removal upon expression in mammalian cells and thus, in someembodiments, such a variant does not contain further amino acidmodifications.

In a further embodiment, said heavy chain comprises two or more chargedamino acid residues in the C-terminal region, such as two, three, fouror five charged amino acid residues, preferably having either all apositive charge or all a negative charge, optionally comprising furtheramino acid modifications C-terminally of said charged residues, e.g. aproline residue situated C-terminally of said charged residues.

In some embodiments, the specified amino acid modifications are theresult of amino acid substitutions. In other embodiments, the specifiedamino acid modifications are the result of C-terminal amino acidadditions.

In one embodiment, said heavy chain comprises a C-terminal sequenceselected from pro-gly-glu (PGE), pro-gly-lys-pro (PGKP; SEQ ID NO:6),pro-gly-lys-lys-pro (PGKKP; SEQ ID NO:7), and pro-gly-lys-lys-lys-pro(PGKKKP; SEQ ID NO:8).

In one embodiment, said antibody is a human antibody, preferably a humanIgG1 or a human IgG3 antibody.

In another embodiment, said antibody is not conjugated at theC-terminus, e.g. not conjugated to another molecule, such as a toxin orlabel. In a further embodiment, said antibody is not conjugated at theC-terminus, but is conjugated at another site of the molecule, e.g. theantibody may, at the other site, be linked to a compound selected fromthe group consisting of: a toxin (including a radioisotope) a prodrug ora drug. Such compounds may make killing of target cells more effective,e.g. in cancer therapy. The resulting antibody is thus animmunoconjugate.

In a further aspect, the invention relates to an antibody of theinvention as described above for use as a medicament, in particular foruse as a medicament for the treatment of diseases or disorders, whereinsilencing (i.e. inhibition) of a target antigen is desired, withoutkilling of the target cell. Examples of such diseases and disordersinclude autoimmune diseases and inflammation.

In a further aspect, the invention relates to a nucleotide constructencoding a heavy chain having a charged amino acid residue in theC-terminal region, wherein said charged amino acid residue is notsusceptible to proteolytic removal. In a further embodiment, saidnucleotide construct has any one or more of the further featuresdescribed above for the antibody of the invention.

In a further aspect, the invention relates to a host cell capable ofproducing an antibody variant of the invention. Such a host cell may bea cell that has been transformed or transfected with a nucleotideconstruct of the invention or a host cell which has been geneticallymodified to eliminate the activity of carboxypeptidases capable ofremoving C-terminal lysines from heavy chains. Examples of host cellsinclude CHO, HEK-293, PER.C6, NSO and Sp2/0.

In an even further aspect, the invention relates to a method forproducing an antibody variant according to the invention comprisingculturing a host cell according to the invention and recovering saidantibody from the cell culture. In one embodiment, said host cell is aCHO or HEK cell comprising a nucleotide construct encoding a heavy chainwith a negatively-charged amino acid residue at the C-terminus or aproline residue preceded by a positively-charged amino acid residue atthe C-terminus.

In one aspect, the invention relates to a method for decreasing theability of an antibody to mediate complement-dependent cytotoxicity,said method comprising the step of adding at least one charged aminoacid residue, a proline residue, or both to the C-terminal region of theheavy-chains of an antibody. In one embodiment, a positively chargedamino acid is inserted N-terminal to a proline residue already presentin the C-terminal region. In one embodiment, a proline residue is addedC-terminal to a lysine residue already present in the C-terminal region.In one embodiment, a positively charged amino acid and a proline isadded to the C-terminal, wherein the positively charged amino acid isadded N-terminal to the proline residue. In such embodiments, thepositively charged amino acid can be selected from lysine, arginine andhistidine, such as lysine or arginine, such as lysine.

In one embodiment, a positively charged amino acid is added to theC-terminal region. In such an embodiment, the positively charged aminoacid can be selected from glutamic acid and aspartic acid.

In one embodiment, the method comprises adding one or more furthercharged amino acids to the C-terminal region.

As explained above, in a further aspect, the invention relates to anantibody mixture comprising a subpopulation of antibody variantmolecules comprising heavy chains having a positively-charged amino acidresidue in the C-terminal region and a subpopulation of antibody variantmolecules comprising heavy chains having a negatively-charged amino acidresidue C-terminal region, wherein said positively-charged andnegatively-charged amino acid residues are not susceptible toproteolytic removal.

Without being bound any specific theory, it is hypothesized thatelectrostatic interactions between oppositely-charged residues reinforceintermolecular antibody interactions and thus facilitate the activationof the complement system.

In one embodiment, each of said subpopulations constitutes at least 10%,such as at least 20% of said mixture, e.g. at least 30% of said mixture.

In another embodiment, the antibody molecules within each of the twosubpopulations comprise two identical heavy chains. In a further aspect,the invention relates to the antibody mixture according to the inventionfor use as a medicament, preferably for use in the treatment of cancer.

As explained above, in a further aspect, the invention relates to anantibody comprising a heavy chain having a variant C-terminal region,said variant C-terminal region comprising one or more amino acidmodifications that favour intermolecular C-terminal interactions betweenantibody molecules. Without being bound by any specific theory, it isbelieved that strengthened intermolecular interactions between antibodymolecules facilitate the activation of the complement system and thusresult in an increased ability to mediate CDC compared to an antibodyproduct lacking said amino acid modifications.

In one embodiment, said antibody comprises two non-identical heavychains. In a particularly interesting embodiment hereof, one heavy chainof the antibody comprises a negatively-charged amino acid residue in theC-terminal region and the other heavy chain of the same antibodycomprises a positively-charged charged amino acid residue in theC-terminal region, wherein said charged amino acid residues are notsusceptible to proteolytic removal. Such an antibody will comprise apositively-charged heavy chain and a negatively-charged heavy chain and,thus, through alignment of oppositely-charged heavy chains betweenantibody molecules, form a multimeric complex containing strongintermolecular antibody-antibody interactions favoring CDC activation.

In one embodiment, such an antibody is a monospecific antibody. Inanother embodiment, it is a bispecific antibody.

In a further aspect, the invention relates to the above-describedantibody of the invention for use as a medicament, preferably for use inthe treatment of cancer.

In an even further aspect, the invention relates to a method forproducing such an antibody comprising culturing a host cell comprisingnucleotide constructs encoding said two different heavy chains andrecovering said antibody from the cell culture of said host cell. Such ahost cell may e.g. be a CHO or HEK cell.

As described above, the present invention inter alia relates tomodifications of antibodies either to remove or add and/or protectcharged amino acid residues at the C-terminus of the heavy chain.Antibodies to be used as starting material of the present inventionbefore modification may e.g. be produced by the hybridoma method firstdescribed by Kohler et al., Nature 256, 495 (1975), or may be producedby recombinant DNA methods. Monoclonal antibodies may also be isolatedfrom phage antibody libraries using the techniques described in, forexample, Clackson et al., Nature 352, 624 628 (1991) and Marks et al.,J. Mol. Biol. 222, 581 597 (1991). Monoclonal antibodies may be obtainedfrom any suitable source. Thus, for example, monoclonal antibodies maybe obtained from hybridomas prepared from murine splenic B cellsobtained from mice immunized with an antigen of interest, for instancein the form of cells expressing the antigen on the surface, or a nucleicacid encoding an antigen of interest. Monoclonal antibodies may also beobtained from hybridomas derived from antibody-expressing cells ofimmunized humans or non-human mammals such as rats, dogs, primates, etc.

Antibodies to be used as starting material of the present invention maybe e.g. chimeric or humanized antibodies. In another embodiment, theantibody is a human antibody. Human monoclonal antibodies may begenerated using transgenic or transchromosomal mice, e.g. HuMAb Mice®,carrying parts of the human immune system rather than the mouse system.The HuMAb Mouse® contains a human immunoglobulin gene minilocus thatencodes unrearranged human heavy (u and y) and k light chainimmunoglobulin sequences, together with targeted mutations thatinactivate the endogenous u and k chain loci (Lonberg, N. et al., Nature368, 856 859 (1994)). Accordingly, the mice exhibit reduced expressionof mouse IgM or k and in response to immunization, the introduced humanheavy and light chain transgenes undergo class switching and somaticmutation to generate high affinity human IgG,κ monoclonal antibodies(Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. Handbook ofExperimental Pharmacology 113, 49 101 (1994), Lonberg, N. and Huszar,D., Intern. Rev. Immunol. Vol. 13 65 93 (1995) and Harding, F. andLonberg, N. Ann. N.Y. Acad. Sci 764 536 546 (1995)). The preparation ofHuMAb Mice® is described in detail in Taylor, L. et al., Nucleic AcidsResearch 20, 6287 6295 (1992), Chen, J. et al., International Immunology5, 647 656 (1993), Tuaillon et al., J. Immunol. 152, 2912 2920 (1994),Taylor, L. et al., International Immunology 6, 579 591 (1994), Fishwild,D. et al., Nature Biotechnology 14, 845 851 (1996). See also U.S. Pat.Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397,5,661,016, 5,814,318, 5,874,299, 5,770,429, 5,545,807, WO 98/24884, WO94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187.Splenocytes from these transgenic mice may be used to generatehybridomas that secrete human monoclonal antibodies according to wellknown techniques.

Further, human antibodies of the present invention or antibodies of thepresent invention from other species may be identified throughdisplay-type technologies, including, without limitation, phage display,retroviral display, ribosomal display, mammalian display, and othertechniques, using techniques well known in the art, and the resultingmolecules may be subjected to additional maturation, such as affinitymaturation, as such techniques are well known in the art.

Target Diseases and Antigens

Antibodies of the invention, lacking C-terminal lysine or other chargedamino acid residues, may be used for many different purposes, such aspurposes where increased cell killing via CDC is desired. Examples ofsuitable diseases include, without limitation, various forms of cancer.Examples of suitable antigen targets for such antibodies include,without limitation, tumor erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4,MUC-1, CD4, CD19, CD20, CD25, CD32, CD37, CD38, CD74, CD138, CXCR5,c-Met, HERV-envelope protein, periostin, Bigh3, SPARC, BCR, CD79,EGFrvIII, IGFr, L1-CAM, AXL, Tissue Factor (TF), EpCAM and MRP3.Preferred antigens include CD20, HER2, EGFR, CD38, IGFR, CD25 and CD32.Exemplary antibodies include HuMAb 7D8, HuMAb 2F2 (described inWO2004035607) and HuMAb 005 (described in WO 2006099875).

Antibodies of the invention containing C-terminal lysine or othercharged amino acid residues may, for example, be used in applicationswhere CDC is not favored as a mode-of-action, e.g. in the treatment ofautoimmune disease or inflammation. Examples of suitable antigen targetsinclude, without limitation TNF-alpha, IL-1, VEGF, IL-6 and IL-8.

Compositions and Uses

In a further main aspect, the invention relates to a pharmaceuticalcomposition comprising an antibody according to the invention asdescribed herein and a pharmaceutically-acceptable carrier.

The pharmaceutical compositions may be formulated in accordance withconventional techniques such as those disclosed in Remington: TheScience and Practice of Pharmacy, 19th Edition, Gennaro, Ed., MackPublishing Co., Easton, Pa., 1995. A pharmaceutical composition of thepresent invention may e.g. include diluents, fillers, salts, buffers,detergents (e.g., a non-ionic detergent, such as Tween® 20 or Tween®80), stabilizers (e.g., sugars or protein-free amino acids),preservatives, isotonicity agents, antioxidants, tissue fixatives,solubilizers, and/or other materials suitable for inclusion in apharmaceutical composition. Examples of suitable aqueous and nonaqueouscarriers which may be employed in the pharmaceutical compositions of thepresent invention include water, saline, phosphate-buffered saline,ethanol, dextrose, polyols (such as glycerol, propylene glycol,polyethylene glycol).

The pharmaceutical composition may be administered by any suitable routeand mode. In one embodiment, a pharmaceutical composition of the presentinvention is administered parenterally. “Administered parenterally” asused herein means modes of administration other than enteral and topicaladministration, usually by injection, and include epidermal,intravenous, intramuscular, intra-arterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal,intratendinous, transtracheal, subcutaneous, subcuticular,intra-articular, subcapsular, subarachnoid, intraspinal, intracranial,intrathoracic, epidural and intrasternal injection and infusion.

The efficient dosages and the dosage regimens for the antibody depend onthe disease or condition to be treated and may be determined by thepersons skilled in the art. An exemplary, non-limiting range for atherapeutically effective amount of an antibody of the present inventionis about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, aboutsuch as 0.3, about 1, about 3, about 5, or about 8 mg/kg.

Antibodies, of the present invention may also be administered incombination therapy, i.e., combined with other therapeutic agentsrelevant for the disease or condition to be treated. Accordingly, in oneembodiment, the antibody-containing medicament is for combination withone or more further therapeutic agents, such as a cytotoxic,chemotherapeutic or anti-angiogenic agents. Such combined administrationmay be simultaneous, separate or sequential. In a further embodiment,the present invention provides a method for treating or preventingdisease, such as cancer, which method comprises administration to asubject in need thereof of a therapeutically effective amount of anantibody of the present invention, in combination with radiotherapyand/or surgery.

EXAMPLES Example 1: Cation Exchange Chromatography (CIEX) Profiles,Capillary Isoelectric Focusing (cIEF) and Sodium DodecylsulphatePolyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis of Anti-CD20 andAnti-CD38 C-terminal Lysine Variants

Anti-CD20 antibody (2F2, described in WO 2004035607 (Genmab)) andanti-CD38 antibody (005, described in WO 2006099875 (Genmab)) wereisolated from hybridoma supernatants and subjected to preparative cationexchange chromatography (CIEX). CIEX was performed on an AKTA Purifiersystem using a ProPac® WCX 10 (9 mm×250 mm) preparative column. Mobilephases A and B were 10 mM sodium phosphate (pH 7.2) and 25 mM sodiumchloride in 10 mM sodium phosphate (pH 7.0). Antibodies were dialyzedO/N prior to injection. Linear gradients from 0% to 12% B in 60 min(anti-CD20) and from 8% to 13% B in 25 min (anti-CD38) were used. Flowrate was 4 mL/min for both antibody separations, and concentrations weredetermined by absorbance at 280 nm. For each antibody, six consecutiveinjections were performed and the individual K0, K1 and K2 isoforms(containing zero, one or two heavy-chain C-terminal lysines perantibody) were pooled, concentrated (Sartorius, Vivaspin®, 10,000 DaMWCO) and separation buffer was exchanged for PBS buffer. Pooledfractions were biochemically analyzed by cIEF and SDS PAGE withouttreatment or after carboxypeptidase B (CPB) treatment (500 μL [450μg/mL] antibody in 20 mM sodium phosphate [pH 7.2] was mixed with 10 μLof 0.05 IU/μL CPB [Calbiochem] and incubated at 37° C. for 4 h). Sampleswere stored at −80° C. until further use. For CIEF analysis, antibodysamples were diluted to 5 μg/mL and 20 μL was loaded onto a precast cIEFFocusGel 6-11 24S (ETC, Kirchentellinsfurt, Germany) with high pI Kitand pH 7.65 and 10.0 markers (Amersham, Piscataway, USA). Gels werepre-focused at 500 V, 30 mA and 10 W for 30 min, followed by focusing at1500 V, 18 mA and 20 W for 90 min and 2000 V, 15 mA and 25 W for 30 min.Gels were fixed in 20% (w/v) trifluoroacetic acid at 30° C. for 45 min.Detection of the bands was performed using ammoniacal silver stainingprocedure recommended by the FocusGel supplier. cIEF gels were digitallyimaged using the GeneGenius Imaging System (Synoptics, Cambridge, UK).All other reagents and devices used for cIEF were obtained from GEHealthcare (Uppsala, Sweden). Non-reduced SDS-PAGE was performed on4-12% NuPAGE™ Bis-Tris SDS-PAGE gels (Invitrogen, Breda, TheNetherlands). SDS-PAGE gels were digitally imaged using the GeneGeniusImaging System (Synoptics, Cambridge, UK).

FIG. 1 shows the CIEX profiles for anti-CD20 (a) and anti-CD38 (b)antibodies. cIEF analysis (FIGS. 1 c and 1 d ) clearly shows that threeIgG charge variants, containing zero, one or two C-terminal lysines perIgG molecule (K0, K1 and K2) are present in unfractionated anti-CD20 andanti-CD38 antibody preparations. Variants could be separated by CIEX asshown in the cIEF profiles of the collected isoforms. SDS PAGE analysis(FIGS. 1 e and 1 f ) shows that structural integrity was maintainedafter CIEX fractionation. Only the K0 isoform was present aftercarboxypeptidase B (CPB) treatment of unfractionated anti-CD20 andanti-CD38 preparations, as well as in collected isoforms (FIGS. 1 c and1 d ).

Example 2: Binding Capacity and Induction of Complement-MediatedCytotoxicity (CDC) by C-Terminal Lysine Isoforms of Anti-CD20 andAnti-CD38 Antibodies

Antibody preparations and collected isoforms, with or withoutcarboxypeptidase B (CPB) treatment, were obtained by preparative CIEX asdescribed supra. Binding of antibody samples to Daudi cells, whichexpress both CD20 and CD38, was analyzed by FACS analysis. 10⁵ cellswere incubated in 50 μL in polystyrene 96-well round bottom plates(Greiner bio-one 650101) with serial dilutions of antibody preparations,ranging from 0.04 μg/mL to 10 μg/mL, in RPMI1640/0.2% BSA at 4° C. for30 min. After washing twice in RPM1/0.2% BSA, cells were incubated withfluorescein isothiocyanate (FITC)-conjugated rabbit anti-human IgG(F0056, Dako, Glostrup, Denmark) at 4° C. for 30 min. Cells were washedtwice in RPM1/0.2% BSA, resuspended in RPM1/0.2% BSA, and analyzed on aFACS Calibur™ (BD Biosciences). Binding curves were analyzed usingnon-linear regression (sigmoidal dose response with variable slope)using GraphPad Prism V5.01 software (GraphPad Software, San Diego,Calif., USA). To test induction of CDC, Daudi cells (2×10⁶ cells/mL)were incubated with serial dilutions of antibody preparations at RT for15 min. Normal human serum (NHS; M0008, Sanquin, Amsterdam, TheNetherlands) was added as a source of complement (final concentration20% [v/v]). After incubation in round-bottom 96-well plates (Nunc™,Rochester, N.Y.) at 37° C. for 45 min, the reaction was stopped byplacing the samples on ice. Cell lysis was determined by FACS analysisusing propidium iodide (PI, Sigma Aldrich, Zwijndrecht, The Netherlands)staining method. % lysis was determined as follows: % lysis=(number ofPI pos cells/total number of cells)×100%.

TABLE 1 Induction of CDC by anti-CD20 and anti-CD38 antibodies. Datashown are EC₅₀ values [μg/mL] for induction of CDC of Daudi cells byunfractionated and collected K2 isoforms, with and withoutcarboxypeptidase B (CPB) treatment, of anti-CD20 and anti-CD38antibodies. Unfract. Unfract. + CPB K2 K2 + CPB anti-CD20 0.84 0.82 1.81.0 anti-CD38 0.12 0.13 0.20 0.16

FIG. 2 upper left panel shows that binding to Daudi cells ofunfractionated and collected K2 isoforms of anti-CD20 antibody with andwithout carboxypeptidase B treatment are comparable. The upper rightpanel shows that the same is true for anti-CD38. The lower left paneland Table 1 show that induction of CDC is less efficient (˜factor 2less) for the collected K2 isoforms of anti-CD20 antibody. Capacity toinduce CDC was restored after carboxypeptidase B treatment of thecollected K2 isoforms of anti-CD20. The lower right panel and Table 1show that the same is true for anti-CD38 antibody. Carboxypeptidase Btreatment partially restored the capacity to induce CDC of the K2isoforms. These data suggest that the presence of C terminal lysinesnegatively influences the capacity of an antibody to induce CDC.

Carboxypeptidase B treatment did not affect the capacity of eitherunfractionated anti-CD20 or anti-CD38 antibody to induce CDC. Theunfractionated antibody preparations do contain C-terminal lysineisoforms (K1 and K2), as shown in FIG. 1 . However, the fraction ofC-terminal lysine containing isoforms in the unfractionated antibodypreparations is probably too small to influence the capacity to induceCDC.

Example 3: Efficacy of C1q Utilization by Unfractionated and CollectedK2 Isoforms of Anti-CD38 Antibody

Antibody preparations and collected K2 isoforms, with or withoutcarboxypeptidase B (CPB) treatment, were obtained as described supra. Totest efficacy of C1q utilization, Daudi cells (2×10⁶ cells/mL) wereincubated in RPMI1640 medium, supplemented with 10% fetal bovine serum,with a fixed concentration (10 μg/mL) of antibody preparations at RT for15 min. C1q-depleted serum (Quidel, San Diego, Calif.) supplemented with1 mM MgCl₂ and 1 mM CaCl₂ and low concentrations of C1q (ComplementTechnologies, Tyler, Tex.) was added as a source of complement (finalconcentration 50% [v/v]). The CDC assay was performed as describedsupra.

TABLE 2 Efficacy of C1q utilization by unfractionated and collected K2isoforms of anti-CD38 antibody. Data shown are EC₅₀ values [μg/mL] forC1q requirement for induction of CDC of Daudi cells by unfractionatedand collected K2 isoforms, with and without CPB treatment, of anti-CD38antibody. Unfract. Unfract. + CPB K2 K2 + CPB anti-CD38 1.3 0.8 4.8 0.7

FIG. 3 and Table 2 show that the amount of C1q required to induce CDC ofDaudi cells was highly increased (>3.5×) for the collected K2 isoformsof anti-CD38. Efficacy of C1q use was completely restored bycarboxypeptidase B treatment. Carboxypeptidase B treatment ofunfractionated anti-CD38 antibody only slightly (<2×) improved theefficacy of C1q utilization.

Example 4: Binding and CDC Induction by HEK-Produced Anti-CD38 Antibodyand Mutants Containing One, Two or Three C-Terminal Lysines or aC-Terminal Glutamic Acid Per Heavy Chain

To further verify that indeed the presence of C-terminal lysines wasresponsible for the decreased efficacy of CDC induction by the collectedK2 isoform compared with the unfractionated antibody preparation,mutants were constructed containing zero, one, two or three C-terminallysines in each heavy chain. Furthermore, a mutant with a negativecharge at the C-terminus (glutamic acid; E) was constructed. Mutants aredescribed in the table below. Amino acid P445 in EU-numberingcorresponds to the proline at position 328 in SEQ ID NOS:1 and 5 (IgG1m(za) and IgG1m(f) allotype Fc-sequences, respectively).

TABLE 3 Mutants of anti-CD38 antibody. pI values were determined by cIEFanalysis (FIG. 4a). C-terminal sequence pI value of (using EU numbering)Abbreviation Charge mutated protein 445-PGE-447 E2 − 8.2 445-PG-446 K0 08.5 445-PGKP-448 K2 + 8.8 445-PGKKP-449 K4 + + 9.0 445-PGKKKP-450K6 + + + 9.1

Mammalian expression vectors for the expression of anti-CD38 antibody005 (described in WO 2006099875 (Genmab)) were constructed by cloningthe coding regions for the human IgG1 (allotype f) heavy and the kappalight chain of the antibody in pcDNA3.3 (Invitrogen). PCR was used tointroduce the C-terminal heavy-chain extensions -KP, -KKP, -KKKP and -Ein the heavy-chain expression vector. Proline was introduced to preventcleavage of the added C-terminal lysine(s). All anti-CD38 antibodymutants were produced, under serum-free conditions, using Freestyle™ Mmedium (Invitrogen, Carlsbad, Calif.) by transiently co-transfectingrelevant heavy and light chain expression vectors in HEK293F cells(Invitrogen) using 293Fectin™ (Invitrogen), according to themanufacturer's instructions. Antibodies were purified by Protein Aaffinity chromatography (MabSelect™ SuRe™, GE Healthcare, Uppsala,Sweden), dialyzed O/N to PBS and filter-sterilized over 0.2 μM dead-endfilters. Concentrations of purified C-terminal IgG1 variants weredetermined by absorbance at 280 nm. Anti-CD38 antibody and anti-CD38antibody mutants were analyzed by CIEF, binding to Daudi cells wasanalyzed by FACS analysis and induction of CDC was tested in a CDC assaywith Daudi cells, all assays were performed as described supra.

TABLE 4 CDC induction by anti-CD38 antibody and mutants. anti-CD38 K0 K2K4 K6 E2 EC₅₀ 0.21 0.21 0.29 N.D.^(a) 0.28 0.26 Max. lysis 97 95 65 9 2526 ^(a)could not be determined. Data shown are EC₅₀ values (μg/mL) andpercentage lysis induced at the highest concentration of antibody tested(4 μg/mL).

FIG. 4 a shows that all antibody mutants migrated at the pI calculatedbased on the amino acids introduced, indicating that the mutants arestable and that the additional C-terminal amino acids were not cleaved.FIG. 4 b shows that binding of anti-CD38 antibody to Daudi cells was notaffected by the introduction of C-terminal lysines or the glutamic acidand was comparable for all mutants. FIG. 4 c and Table 4 show thatinduction of CDC of Daudi cells was similar for anti-CD38 antibody andPG mutant. Introduction of one C-terminal lysine, which has a positivecharge, per heavy chain, decreased the capacity to induce CDC, reflectedin a lower percentage of maximal lysis (˜30% decrease) induced.Introduction of two C-terminal lysines (KK) per heavy chain totallyabolished the capacity to induce CDC. The mutant containing three Cterminal lysines per heavy chain was slightly more efficient ininduction of CDC than the mutant containing two C-terminal lysines. Themutant containing C-terminal glutamic acid was inefficient in inducingCDC as well.

Example 5: CDC Induction by Mixtures of Anti-CD38 Antibody Mutants

To investigate whether charge repulsion between IgG molecules isresponsible for the reduced CDC activity of anti-CD38 mutants carryinglysine or glutamic acid at the C-terminus, mutants with differentcharges at the C-terminus were mixed. The CDC assay was performed asdescribed supra.

FIG. 5 a shows that adding K4 mutant to K0, while keeping the total IgGconcentration constant, decreased the efficacy of CDC induction,compared with that of K0 alone. FIG. 5 b shows that the same effect wasobserved for addition of the E2 mutant. These experiments are in goodagreement with our previous findings and confirm that mutants ofanti-CD38 with charged residues at their C-terminus have stronglyreduced CDC activity. FIG. 5 c shows that mixing the negatively chargedE2 mutant with the positively charged K4 mutant almost fully restoredefficacy of CDC induction, to the level of CDC induced by K0. Similarresults were obtained for a mixture of E2 mutant and K2 mutant (data notshown). FIG. 5 d shows that maximal lysis was obtained at an E2:K4 ratioof approximately 7:3.

The data suggest that interaction between different IgG molecules, andmore specifically Fc-Fc interaction, plays an important role in theinduction of CDC.

Example 6: Identification of IgG1 Mutations Stimulating Fc:FcInteraction-Mediated Antibody Oligomerization Detected by a CDC Assay

A library of mutations was generated, including those focused at theC-terminal of the CH3 domain. Mutations were introduced into the IgG1Fc-region of anti-CD38 antibody 005 using the Quikchange® site-directedmutagenesis kit (Stratagene, US). Briefly, for each desired mutationposition, a forward and a reverse primer encoding a degenerate codon atthe desired location were used to replicate full length plasmid DNAtemplate. The resulting DNA mixtures were digested using DpnI to removesource plasmid DNA and used to transform E. coli. Resulting colonieswere pooled, cultured and plasmid DNA was isolated from these pools andretransformed into E. coli to obtain clonal colonies. Mutant plasmid DNAisolated from resulting colonies was checked by DNA sequencing (LGCgenomics, Berlin, Germany). Expression cassettes were amplified fromplasmid DNA by PCR and DNA mixtures containing both a mutant heavy and awild type light chain of anti-CD38 antibody 005 were transientlytransfected to Freestyle™ HEK293F cells (Invitrogen, US) using293Fectin™ (Invitrogen, US) essentially as described by themanufacturer. Supernatants of transfected cells containing antibodymutants were collected.

CDC assays were performed as follows. 0.1×10⁶ Daudi or Wien 133 cellswere pre-incubated in round-bottom 96-well plates with 1.0 μg/mLunpurified antibodies in a total volume of 100 μL for 15 min on a shakerat RT. Next, 30 μL normal human serum was added as a source ofcomplement (30% final concentration) and incubated in a 37° C. incubatorfor 45 min. The reaction was stopped by putting the plates on ice. 10 μLpropidium iodide was added and cell lysis was determined by FACS.

Mutations in the CH3 C-terminal region incorporated in the anti-CD38antibody were tested for their ability to induce CDC of Daudi cells. Thelytic effect of the mutant antibody was compared to that of wild typeantibody, for which lysis was set to 100%. The cut-off for inhibitionwas set to 66% lysis. The results are shown in Table 5.

Mutations incorporated in the anti-CD38 antibody 005 were tested fortheir ability to enhance oligomerization as determined by CDC on Wien133 cells. Wild type antibody is not able to induce CDC on Wien 133cells. Mutants displaying ≥0% cell lysis were scored as enhancing. Theresults are shown in Table 6.

Table 5 shows that introduction of a negative charge at the C-terminusfor mutations P445D and P445E, which decrease CDC when compared to theIgG1 wildtype, in line with expectations. Similarly, introduction of apositive charge, i.e. P445H and P445K, decreases CDC in line withexpectations. The mutation K447I, however, apparently decreases CDC,whereas removal of the C-terminally positively charged K was expectingto have a positive impact on CDC. In addition, replacement of G446 andK447 with arginine seems to have a small positive effect on CDC,contrary to expectations. However, this assumes that the C-terminalarginine is, in fact, present on the mutants. In both cases, thearginine mutants may actually have been C-terminally cleaved, resultingin mutants lacking C-terminal charge (the theoretic c-terminal sequenceof the K447R mutant is PGR, which may be cleaved to PG, and thetheoretic c-terminal sequence of the G446R mutant is PRK, which may becleaved to P).

TABLE 5 CDC induction by mutants - Daudi cells. Residue A C D E F G H IK L M N P Q R S T V W Y P445 75 94 25 22 95 60 41 43 77 65 84 G446 72 5663 83 84 57 88 K447 92 28 70 71 80 77 96 Percentage lysis of Daudi cellsin the presence of 1.0 ug/ml anti-CD38 antibody point mutants. Wildtypeantibody lysed 66% of cells under these conditions.

TABLE 6 CDC induction by mutants - Wien 133 cells. Residue A C D E F G HI K L M N P Q R S T V W Y P445 3 5 2 2 4 2 4 4 3 2 3 G446 3 4 5 4 7 3 7K447 3 4 3 3 2 2 6 Percentage lysis of Wien 133 cells in the presence of1.0 ug/ml anti-CD38 antibody point mutants. Wildtype antibody lysed 3%of cells under these conditions.

The invention claimed is:
 1. An isolated IgG antibody comprising a heavychain comprising at the C-terminus an amino acid sequence selected fromthe group consisting of: PGE, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:8, wherein said amino acid sequence inhibits proteolytic removal by amammalian protease.
 2. The antibody according to claim 1, furthercomprising a light chain.
 3. The antibody according to claim 1, whereinthe antibody induces complement-dependent cytotoxicity to a lesserextent compared to an antibody lacking said amino acid sequence.
 4. Theantibody of claim 1, wherein the amino acid sequence at the C-terminuscomprises the sequence PGE.
 5. The antibody of claim 1, wherein theamino acid sequence at the C-terminus comprises SEQ ID NO:
 6. 6. Theantibody of claim 1, wherein the amino acid sequence at the C-terminuscomprises SEQ ID NO:
 7. 7. The antibody of claim 1, wherein the aminoacid sequence at the C-terminus comprises SEQ ID NO:
 8. 8. The antibodyof claim 1, wherein, other than said amino acid sequence, the IgGantibody is an isotype selected from the group consisting of: IgG1,IgG2, IgG3, and IgG4.
 9. The antibody of claim 1, wherein the proteaseis selected from a protease of CHO, HEK293, PER.C6, NSO, or Sp2/0 cellorigin.
 10. The antibody of claim 1, wherein the protease is acarboxypeptidase.
 11. A nucleotide construct encoding an IgG heavy chaincomprising at the C-terminus an amino acid sequence selected from thegroup consisting of: PGE, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8,wherein said amino acid sequence inhibits proteolytic removal by amammalian protease.
 12. The nucleotide construct according to claim 11,further encoding a light chain.
 13. The nucleotide construct of claim11, wherein the amino acid sequence at the C-terminus comprises thesequence PGE.
 14. The nucleotide construct of claim 11, wherein theamino acid sequence at the C-terminus comprises SEQ ID NO:
 6. 15. Thenucleotide construct of claim 11, wherein the amino acid sequence at theC-terminus comprises SEQ ID NO:
 7. 16. The nucleotide construct of claim11, wherein the amino acid sequence at the C-terminus comprises SEQ IDNO:
 8. 17. The nucleotide construct of claim 11, wherein, other thansaid amino acid sequence, the IgG antibody is an isotype selected fromthe group consisting of: IgG1, IgG2, IgG3, and IgG4.
 18. The nucleotideconstruct of claim 11, wherein the protease is selected from a proteaseof CHO, HEK293, PER.C6, NSO, or Sp2/0 cell origin.
 19. The nucleotideconstruct of claim 11, wherein the protease is a carboxypeptidase.
 20. Aisolated recombinant host cell which produces the antibody of claim 1,wherein the recombinant cell is genetically engineered to produceglycoproteins having human-like or human glycosylation.
 21. A method forproducing an IgG antibody comprising a heavy chain comprising at theC-terminus an amino acid sequence selected from the group consisting of:PGE, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein said aminoacid sequence inhibits proteolytic removal by a mammalian protease, themethod comprising culturing the host cell according to claim 20 andrecovering said antibody from the cell culture.
 22. The method accordingto claim 21, wherein said host cell is a CHO or HEK cell.
 23. Anisolated IgG antibody comprising a heavy chain comprising at theC-terminus an amino acid sequence selected from the group consisting of:PGE, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, wherein said aminoacid sequence inhibits proteolytic removal by a mammalian protease, andwherein the antibody induces complement-dependent cytotoxicity to alesser extent compared to an antibody lacking said amino acid sequence.